CN110655587A - Preparation method of bletilla striata polysaccharide - Google Patents
Preparation method of bletilla striata polysaccharide Download PDFInfo
- Publication number
- CN110655587A CN110655587A CN201910905352.4A CN201910905352A CN110655587A CN 110655587 A CN110655587 A CN 110655587A CN 201910905352 A CN201910905352 A CN 201910905352A CN 110655587 A CN110655587 A CN 110655587A
- Authority
- CN
- China
- Prior art keywords
- bletilla striata
- preparation
- polysaccharide
- bletilla
- striata polysaccharide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0087—Glucomannans or galactomannans; Tara or tara gum, i.e. D-mannose and D-galactose units, e.g. from Cesalpinia spinosa; Tamarind gum, i.e. D-galactose, D-glucose and D-xylose units, e.g. from Tamarindus indica; Gum Arabic, i.e. L-arabinose, L-rhamnose, D-galactose and D-glucuronic acid units, e.g. from Acacia Senegal or Acacia Seyal; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Sustainable Development (AREA)
- Emergency Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly relates to a preparation method of bletilla striata polysaccharide. The preparation method comprises the following steps: drying rhizoma Bletillae root tuber, pulverizing, and sieving; adding rhizoma bletilla powder into the prepared low molecular alcohol/salt aqueous two-phase system solution, performing microwave-assisted extraction, centrifuging at high speed to separate out lower phase, ultrafiltering, concentrating, desalting, and freeze drying to obtain rhizoma bletilla polysaccharide. Compared with the prior art, the method can effectively shorten the extraction time, improve the extraction purity and reduce the extraction steps, is simple and convenient to operate, and is easy for industrial scale preparation. The prepared bletilla striata polysaccharide has the advantages of complete structure, high polysaccharide yield, high protein removal rate, low toxicity and safety, can prevent damage of a skin barrier, promotes the repair process of the skin barrier damage, and has good anti-allergy effect on sensitive skin with abnormal skin barrier function.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the technical field of biology, and particularly relates to a preparation method of bletilla striata polysaccharide.
[ background of the invention ]
Bletilla striata is a dry tuber of a perennial herb Bletilla striata (Thunb.) Reichb.f. of the family Orchidaceae, is commonly seen in shading grassland or under-forest wet lands, and is continuously grown due to white root color, so that the Bletilla striata is famous and distributed in various places of China. The part of the Chinese pharmacopoeia where bletilla striata is used as a medicine is a tuber, and the main active ingredients of the Chinese pharmacopoeia are bletilla striata polysaccharide, also called bletilla striata gum and bletilla striata mannan. The research shows that the content of the dried bulb tissue is about 40-50%, the polysaccharides mainly comprise beta-1, 4-mannose, beta-1, 4-glucose and beta-1, 6-glucose residues, and belong to neutral heteropolysaccharide, and the main molecular weight span of the bletilla striata polysaccharide is large, and the molecular weight range of the bletilla striata polysaccharide is from tens of thousands to millions. Modern pharmacological research shows that the bletilla striata polysaccharide has biological activities of resisting inflammation, promoting blood coagulation, resisting virus, resisting tumor, resisting oxidation and the like; as a natural polymer material, the bletilla striata polysaccharide has good histocompatibility, biological adhesiveness, biodegradability, no irritation to mucous membrane, abundant resources, low price and easy availability, and has the characteristics of a biological adhesion carrier material.
The existing bletilla striata polysaccharide extraction method comprises the following steps: water extraction, water extraction and alcohol precipitation, continuous countercurrent extraction and enzyme process. The single water extraction method is subjected to high-temperature treatment, and the process often causes that the starch in the bletilla striata medicinal material is dissolved out along with the polysaccharide, so that the prepared bletilla striata polysaccharide contains a large amount of starch; in the preparation process of the enzyme method, although inactive polysaccharides such as starch and the like are not dissolved out, impurities such as protein and the like are introduced in the extraction process, and the protein and the starch are macromolecular structures and are difficult to remove in the later purification process, so that the comprehensive development of the bletilla striata polysaccharide is limited. In addition, after the extraction by the method, one or more methods such as sevage method protein removal, ethanol precipitation, ion exchange chromatography, gel filtration chromatography, affinity adsorption chromatography and the like are combined to purify the bletilla striata polysaccharide, so that the defects of low yield, high reagent consumption, high energy consumption, high price, complicated operation steps and the like exist.
In view of the above, it is desirable for the practitioners to develop an extraction and purification method of bletilla striata polysaccharide, which is suitable for industrial production, and has good purity and high yield. An Aqueous two-phase extraction (ATPS) technique is also called an Aqueous two-phase separation technique, which is attracting attention in recent years, and a very promising novel separation technique, which has been widely applied in the fields of biochemistry, cell biology, biochemical engineering, etc. When the active ingredients in the biological substance or natural product enter the aqueous two-phase system, the active ingredients in the biological substance or natural product are easily selectively distributed between two phases due to the influence of surface properties, electric charge effects, the existence of various forces (hydrophobic bonds, hydrogen bonds, and ionic bonds), and environmental factors (concentration, temperature, pH, and the type and strength of ions). Especially, the low molecular alcohol/salt aqueous two-phase system has the advantages of low price, low toxicity, small viscosity and the like, and can be used for extracting and separating natural products. However, no report is found on the current research on the extraction of bletilla striata polysaccharide by using an aqueous two-phase system. Therefore, the alcohol salt ratio of low-molecular alcohol and different inorganic salts such as potassium salt, sodium salt, ammonium salt and the like and the stability of forming a two-aqueous-phase system are researched, a simple and efficient bletilla striata polysaccharide preparation method is established, and the bletilla striata polysaccharide is used for preventing skin barrier damage and promoting skin barrier damage repair. The method adopts microwave-assisted aqueous two-phase technology to directly introduce rhizoma bletilla polysaccharide extract into water layer, and simultaneously extract impurities such as protein to upper phase or two-phase interface, thereby improving purity of target product and avoiding organic solvent residue in the existing process. The technology is simple and convenient, economical, time-saving, green and efficient, and convenient to amplify.
[ summary of the invention ]
The invention aims to provide a quick and simple preparation method of bletilla striata polysaccharide, and the method has high extraction rate of the bletilla striata polysaccharide and achieves the aim of quickly extracting and purifying a target product.
The invention solves the technical problems through the following technical scheme:
the preparation method of the bletilla striata polysaccharide specifically comprises the following operation steps:
(1) treatment of bletilla striata raw materials: drying rhizoma bletilla tuber at 60-80 deg.C to constant weight, pulverizing, and sieving to obtain 20-80 mesh powder of bletilla striata;
(2) preparing a low molecular alcohol/salt aqueous two-phase system: preparing a low-molecular-weight alcohol and inorganic salt aqueous two-phase system at room temperature, adjusting the pH value of the solution to 7.0-8.0, fully stirring and uniformly mixing to establish an aqueous two-phase system;
(3) performing double-aqueous phase extraction and microwave extraction: adding bletilla striata powder into a double aqueous phase system to prepare an extraction solution, and carrying out microwave treatment on the extraction solution at 100-600W and 30-60 ℃ for 60-180 min; fully extracting the bletilla polysaccharide, standing for 60-100 min, and centrifuging at 8000-12000 rpm at a high speed to separate a bletilla polysaccharide extract phase;
(4) ultrafiltration, concentration and desalination: performing ultrafiltration desalination on the bletilla striata polysaccharide extract phase obtained in the step (3) by using an ultrafiltration membrane with the molecular weight cutoff of 1-10KD to obtain bletilla striata polysaccharide concentrated solution;
(5) and (3) freeze drying: and (4) freeze-drying the bletilla striata polysaccharide concentrated solution obtained in the step (4) to obtain white uniform bletilla striata polysaccharide solid.
In the step (2), the low molecular alcohol is a linear or branched alkyl alcohol having 1 to 5 carbon atoms, and particularly preferably ethanol or isopropanol.
Further, in the step (2), the inorganic salt is a potassium salt, a sodium salt, or an ammonium salt, and particularly preferably potassium carbonate, dipotassium hydrogen phosphate, sodium carbonate, disodium hydrogen phosphate, ammonium sulfate, or ammonium carbonate.
Further, in the step (2), the mass fraction of the inorganic salt is 10% to 40%, and the mass fraction of the low molecular alcohol is 20% to 25%.
Further, in the step (2), the pH value of the aqueous two-phase solution is 7.0-8.0, and particularly preferably is 7.5.
Furthermore, in the step (3), the solid-to-liquid ratio of the bletilla striata powder to the aqueous two-phase solution is 1: 20-1: 100, and particularly preferably 1: 50-1: 80.
Further, in the step (3), the microwave treatment is preferably performed at 400W and 50 ℃ for 120 min.
In the step (4), ultrafiltration desalination is particularly preferably performed by an ultrafiltration membrane with the molecular weight cut-off of 5 KD.
Further, in the step (5), the average molecular weight distribution of the bletilla striata polysaccharide solid is 1-100KD, and particularly preferably 25 KD; the monosaccharide composition molar ratio n (mannose) of the compound is as follows: n (glucose) ═ 4:1 to 1:1, and particularly preferably 2.7:1.
the alcohol salt ratio of low molecular alcohol and different inorganic salts such as potassium salt, sodium salt, ammonium salt and the like and the stability of forming a two-aqueous-phase system are researched, a simple and efficient bletilla striata polysaccharide preparation method is established, and the bletilla striata polysaccharide is used for preventing skin barrier damage and promoting skin barrier damage repair. The method adopts microwave-assisted aqueous two-phase technology to directly introduce rhizoma bletilla polysaccharide extract into water layer, and simultaneously extract impurities such as protein to upper phase or two-phase interface, thereby improving purity of target product and avoiding organic solvent residue in the existing process. The technology is simple and convenient, economical, time-saving, green and efficient, and convenient to amplify.
Compared with the prior art, the invention has the beneficial effects that: 1) by adopting double-aqueous-phase extraction, impurities such as protein and the like can be extracted to an upper phase or a two-phase interface while bletilla striata polysaccharide is extracted to an aqueous phase, so that the purity of a target product is improved; by combining with microwave extraction technology, the extraction rate of bletilla striata polysaccharide can be greatly improved, the extraction period is shortened, and the recovery rate is high. 2) The technology is simple and convenient, economical, time-saving, green and efficient, and convenient to amplify. The prepared bletilla striata polysaccharide has a complete structure, low toxicity and safety, can prevent damage of a skin barrier, promotes the repair process of the damage of the skin barrier, and has a good anti-allergy effect on sensitive skin with abnormal skin barrier functions.
[ description of the drawings ]
The invention will be further described with reference to the following examples with reference to the accompanying drawings.
FIG. 1 is an infrared spectrum of bletilla striata polysaccharide of the present invention.
FIG. 2 is a high performance size exclusion chromatogram (HPSEC-ELSD) of bletilla striata polysaccharides of the present invention.
FIG. 3 is HPLC chromatogram analysis of monosaccharide components of bletilla striata polysaccharides of the present invention.
[ detailed description ] embodiments
The invention relates to a preparation method of bletilla striata polysaccharide, which specifically comprises the following operation steps:
(1) treatment of bletilla striata raw materials: drying rhizoma bletilla tuber at 60-80 deg.C to constant weight, pulverizing the dried tuber by high speed tissue triturator, and sieving to obtain 20-80 mesh powder of bletilla striata;
(2) preparing a low molecular alcohol/salt aqueous two-phase system: preparing a low molecular alcohol and inorganic salt aqueous two-phase system at room temperature, accurately weighing inorganic salt, adding a proper amount of water to prepare a certain concentration, adding a proper amount of low molecular alcohol after dissolution, adjusting the pH value of the solution, fully stirring and uniformly mixing to establish the aqueous two-phase system.
(3) Performing double-aqueous phase extraction and microwave extraction: adding bletilla striata powder into a double aqueous phase system according to a certain solid-liquid ratio to prepare an extraction solution, and carrying out microwave treatment on the extraction solution at the temperature of 30-60 ℃ for 60-180 min under the condition of 100-600W; fully extracting bletilla striata polysaccharide, standing for 60-100 min, centrifuging at 8000-12000 rpm, and separating bletilla striata polysaccharide extract phase.
(4) Ultrafiltration, concentration and desalination: and (4) carrying out ultrafiltration desalination on the bletilla striata polysaccharide extract phase obtained in the step (3) by using an ultrafiltration membrane with the molecular weight cutoff of 1-10KD, and adding water twice during the ultrafiltration desalination to obtain the bletilla striata polysaccharide concentrated solution.
(5) And (3) freeze drying: and (4) freeze-drying the bletilla striata polysaccharide concentrated solution obtained in the step (4) to obtain white uniform bletilla striata polysaccharide solid.
In the step (2), the low molecular alcohol is a C1-C5 linear or branched alkyl alcohol, and particularly preferably one of ethanol and isopropanol.
In the step (2), the inorganic salts are potassium salts, sodium salts and ammonium salts, and particularly preferred is potassium carbonate, dipotassium hydrogen phosphate, sodium carbonate, disodium hydrogen phosphate, ammonium sulfate or ammonium carbonate.
In the step (2), the mass fraction of the inorganic salt is 10-40%, and the mass fraction of the low molecular alcohol is 20-25%.
In the step (2), the pH value of the aqueous two-phase solution is 7.0-8.0, and particularly preferably is 7.5.
In the step (3), the solid-to-liquid ratio of the bletilla striata powder to the aqueous two-phase solution is 1: 20-1: 100, and particularly preferably 1: 50-1: 80.
In the step (3), the microwave treatment is carried out under conditions of 400W, preferably at 50 ℃ for 120 min.
In step (4), ultrafiltration desalting with an ultrafiltration membrane having a molecular weight cut-off of 5kD is particularly preferable.
The content and the extraction rate of the bletilla striata polysaccharide are detected by adopting an anthrone sulfate colorimetric method, the molecular weight of the bletilla striata polysaccharide is detected by adopting a high performance gel permeation chromatography-ELSD (HPGPC), the monosaccharide composition and the proportion are detected by adopting a PMP derivative high performance liquid chromatography, and infrared spectrum analysis is combined, so that the effect of extracting the bletilla striata polysaccharide by combining double water phases with microwave extraction and the product quality characteristics are fully known.
Anthrone-concentrated sulfuric acid colorimetry: and (3) drying the standard grape product in an oven to constant weight, accurately weighing 0.1g to prepare a 1.0mg/mL glucose solution, and diluting by 10 times to obtain a 100 mu g/mL glucose standard solution. Accurately measuring glucose standard solution 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL, adding 0.2% anthrone-concentrated sulfuric acid solution (0.5mL anthrone solution and 4mL concentrated sulfuric acid) into ice water bath, reacting in boiling water bath for 10min, taking out, placing in ice water bath for 10min, detecting light absorption value at wavelength of 625nm, and making standard curve. Measuring the absorbance value of rhizoma bletilla polysaccharide by the same method, determining the quality of rhizoma bletilla polysaccharide according to the drawn standard curve, and calculating polysaccharide content and extraction rate.
The calculation formula of the bletilla polysaccharide content is as follows: weighing rhizoma bletilla polysaccharide (weight ratio of rhizoma bletilla polysaccharide to weight ratio of 100%.
The formula for calculating the extraction rate of the bletilla striata polysaccharide is as follows: the extraction rate (g/g) of the bletilla striata polysaccharide is equal to the mass of the bletilla striata polysaccharide/mass of the bletilla striata powder multiplied by 100%.
The present invention will be further described with reference to the following examples.
Example 1
Drying rhizoma bletilla tuber at 60-80 deg.C to constant weight, pulverizing the dried tuber by high speed tissue triturator, and sieving to obtain 20-80 mesh powder of bletilla striata; according to the design of table 1, a certain mass of sodium carbonate and a certain volume of absolute ethyl alcohol are weighed, ultrapure water is added firstly, stirring is carried out to dissolve inorganic salt, then absolute ethyl alcohol is added, finally water is supplemented to 1000ml of final volume, stirring is carried out fully, and the pH value of the aqueous two-phase solution is adjusted to 7.5. Weighing certain amount of rhizoma bletilla powder, adding into the double water phase system, stirring, microwave treating at 400W and 50 deg.C for 120min to fully separate out rhizoma bletilla polysaccharide, standing for 60min, centrifuging at 10000rpm for 30min, separating rhizoma bletilla polysaccharide extract phase, ultrafiltering and desalting with ultrafiltration membrane with cut-off molecular weight of 5KD, and adding water twice during the process to obtain rhizoma bletilla polysaccharide concentrated solution. Freeze drying the concentrated solution to obtain white uniform rhizoma bletilla polysaccharide solid.
TABLE 1 aqueous two-phase composition and bletilla striata polysaccharide extraction results
Example 2
Drying rhizoma bletilla tuber at 60-80 deg.C to constant weight, pulverizing the dried tuber by high speed tissue triturator, and sieving to obtain 20-80 mesh powder of bletilla striata; according to the design of table 1, a certain mass of disodium hydrogen phosphate and a certain volume of absolute ethyl alcohol are weighed, ultrapure water is added firstly, stirring is carried out to dissolve inorganic salt, then absolute ethyl alcohol is added, water is added finally until the final volume of 1000ml, stirring is carried out fully, and the pH value of the aqueous two-phase solution is adjusted to 7.5. Weighing certain amount of rhizoma bletilla powder, adding into the double water phase system, stirring, microwave treating at 400W and 50 deg.C for 120min to fully separate out rhizoma bletilla polysaccharide, standing for 60min, centrifuging at 10000rpm for 30min, separating rhizoma bletilla polysaccharide extract phase, ultrafiltering and desalting with ultrafiltration membrane with cut-off molecular weight of 5KD, and adding water twice during the process to obtain rhizoma bletilla polysaccharide concentrated solution. Freeze drying the concentrated solution to obtain white uniform rhizoma bletilla polysaccharide solid.
TABLE 2 aqueous two-phase composition and bletilla striata polysaccharide extraction results
Example 3
Drying rhizoma bletilla tuber at 60-80 deg.C to constant weight, pulverizing the dried tuber by high speed tissue triturator, and sieving to obtain 20-80 mesh powder of bletilla striata; according to the design of table 1, a certain mass of sodium carbonate and a certain volume of isopropanol are weighed, ultrapure water is added firstly, stirring is carried out to dissolve inorganic salt, then absolute ethyl alcohol is added, finally water is supplemented to 1000ml of final volume, stirring is carried out fully, and the pH value of the aqueous two-phase solution is adjusted to 7.5. Weighing certain amount of rhizoma bletilla powder, adding into the double water phase system, stirring, microwave treating at 400W and 50 deg.C for 120min to fully separate out rhizoma bletilla polysaccharide, standing for 60min, centrifuging at 10000rpm for 30min, separating rhizoma bletilla polysaccharide extract phase, ultrafiltering and desalting with ultrafiltration membrane with cut-off molecular weight of 5KD, and adding water twice during the process to obtain rhizoma bletilla polysaccharide concentrated solution. Freeze drying the concentrated solution to obtain white uniform rhizoma bletilla polysaccharide solid.
TABLE 3 aqueous two-phase composition and bletilla striata polysaccharide extraction results
Example 4
The molecular weight of the bletilla striata polysaccharide was determined by high performance gel permeation chromatography-ELSD (HPGPC) (see figure 2). The chromatographic conditions are as follows: g-6000 chromatographic column (6.0 × 400mm), the mobile phase is water solution, the mass concentration of the standard substance and the sample is 2 mg.mL < -1 >, the sample amount is 10 μ L, the flow rate is 0.5 mL.min < -1 >, the column temperature is 40 ℃, and the detector is an evaporation light detector ELSD (temperature 110 ℃, gain 2).
And (3) preparing a standard curve: preparing glucose and dextran series standard substances (5, 50, 100, 200 and 500KDa) into solutions with mass concentration of 2 mg.mL < -1 > respectively by using aqueous solutions, sequentially injecting samples from small to large according to molecular mass, and determining retention time Ve. Vt and V0 were calibrated with glucose and T-200 dextran, respectively. The distribution coefficient (KaV) and retention time are related as follows: KaV ═ Ve-V0/Vt-V0; a standard curve was plotted with KaV as the abscissa and lgMw (log of molecular mass) as the ordinate.
And (3) measuring the molecular weight: preparing a sample to be detected into a solution with the mass concentration of 2 mg.mL < -1 > by using an aqueous solution, and determining the retention time Ve. And (4) deducing the molecular weight of the sample according to the quantitative relation of the standard curve. The results are shown in FIG. 2, and the molecular weight of the obtained sample is 23 KD.
Example 5
Taking a dried bletilla striata polysaccharide sample of 2mg, tabletting with KBr, performing infrared spectrum scanning within the range of 4000-400 cm < -1 >, and recording an infrared spectrum chart (see attached figure 1). The results show that the bletilla striata polysaccharide prepared by the invention comprises fingerprint region absorption of glucose and mannose in beta configuration and characteristic acetyl absorption (near 1743 cm-1). According to an IR spectrogram, the bletilla striata polysaccharide has a characteristic absorption peak of polysaccharide, and the absorption peak at 3600-3200 cm < -1 > (3372.27cm < -1 >) is stretching vibration of O-H; 3000-2750 cm-1 (2887.97cm-1) has 2 absorption peaks, which indicate the presence of saccharide-CH2C-H symmetric and antisymmetric telescopic vibration; an absorption at 1742.31cm-1 indicates modification with an acyl group. Wave numbers of 811.46cm-1 and 871.05cm-1 are characteristic absorptions of mannose residues, and at wave number of 920cm-1, an absorption is observed, indicating the presence of a glucopyranose residue. The characteristic absorption peak of the glycosidic bond is no obvious absorption at 840cm-1, which indicates that no alpha type glycosidic bond exists, while the absorption peak at 871.05cm-1 indicates that the glycosidic bond configuration is beta type.
Example 6
Analysis of bletilla striata polysaccharide monosaccharide composition and ratio (example 1)
Measuring monosaccharide composition and proportion by PMP derivative high performance liquid chromatography
Chromatographic conditions are as follows: agilent HPLC system, column kromasil C18(4.6 mm. times.150 mm,5 μ M), mobile phase 82.0% PBS (0.05M, pH7.0) and 18.0% acetonitrile (v/v), flow rate of 1.0mL min-1, sample size of 10 μ L, detection wavelength of 254 nm.
Precisely weighing 2mg of bletilla striata polysaccharide sample, respectively adding 0.5ml of 2M trifluoroacetic acid, hydrolyzing at 120 ℃ for 2 hours, adding a small amount of methanol, evaporating to dryness in a water bath at 45 ℃, and repeating for 3-5 times until the trifluoroacetic acid is completely evaporated. And adding 1.0ml of purified water into the dried sample obtained after complete acid hydrolysis for dissolving, taking 100ul of sample solution for derivatization operation of 1-phenyl-3-methyl-5-pyrazolone (PMP), and diluting by 20 times for detection.
The HPLC chromatogram of monosaccharide of the product obtained by hydrolyzing the monosaccharide mixed reference substance and the purified component of rhizoma bletilla polysaccharide No. 5 sample with trifluoroacetic acid is shown in figure 3, and the retention time and peak area of the appearance sequence are shown in Table 3. The results show that the bletilla striata polysaccharide No. 5 sample mainly comprises mannose and glucose, and the monosaccharide comprises a molar ratio n (mannose): n (glucose) ═ 2.7: 1.0.
Example 7
In vitro experiments discuss the protective effect of bletilla striata polysaccharide on skin fibroblasts under ultraviolet irradiation.
The experimental method comprises the following steps: culturing HSf human skin fibroblast strain in 10% FBS DMEM medium, setting normal control group, ultraviolet irradiation group, and ultraviolet irradiation + bletilla striata polysaccharide (20ug/ml) group, irradiating with SUV-1000 for 7 days for 30min per day with irradiation dose of 1J/cm2Total cumulative exposure of 7J/cm2And observing the changes of fibroblast senescence markers beta-galactosyl glycosidase and collagen degrading enzyme MMP-1 under ultraviolet irradiation by using a cell immunohistochemical method.
TABLE 4 changes in beta-galactosidases and MMP-1 (. about.P <0.05)
The in vitro test indicates that the bletilla striata polysaccharide has obvious protective effect on skin fibroblast aging and collagen degradation caused by ultraviolet rays. The monosaccharide composition, the connection mode, the glycosidic bond type and the branching degree all make great contribution to the application of the bletilla striata polysaccharide, the bletilla striata polysaccharide is different from other plant polysaccharides, the monosaccharide composition comprises mannose and glucose, the monosaccharide composition proportion is different, and the difference of the bioactivity is reflected. When the monosaccharide composition molar ratio n (mannose): the effect is obvious when n (glucose) is 2.7: 1.0.
Example 8
The repairing effect of bletilla striata polysaccharide on skin barrier damage is evaluated by testing the water content of the horny layer and the transdermal water loss rate of the skin. The subjects were selected as individuals without a history of skin disease and cosmetic allergy between 20 and 40 years of age on a voluntary basis. The specific method is that the bletilla striata polysaccharide emulsion of the invention is applied to the skin of the symmetrical part of the same volunteer in a random double-blind mode, 10 subjects are randomly selected for each sample, about 20g of the sample is uniformly smeared on the face every time, other moisturizing products are not used in the test period, the positions of test points are marked by a positioning film, and the test points are respectively used once in the morning and evening every day for 4 weeks. The degree of epidermal water loss (TEWL) and the degree of hydration of the horny layer of the skin were measured at week 0, week 2 and week 4 using a multifunctional skin tester model MC760 from CK, Germany. Tests prove that the bletilla striata polysaccharide prepared by the method has obvious effects of repairing skin barrier and moisturizing. When the monosaccharide composition molar ratio n (mannose): the effect is obvious when n (glucose) is 2.7: 1.0.
Table 5 TEWL and change in the degree of hydration of the stratum corneum (. P <0.05)
Although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.
Claims (9)
1. A preparation method of bletilla striata polysaccharide is characterized by comprising the following steps: the method comprises the following operation steps:
(1) treatment of bletilla striata raw materials: drying rhizoma bletilla tuber at 60-80 deg.C to constant weight, pulverizing, and sieving to obtain 20-80 mesh powder of bletilla striata;
(2) preparing a low molecular alcohol/salt aqueous two-phase system: preparing a low-molecular-weight alcohol and inorganic salt aqueous two-phase system at room temperature, adjusting the pH value of the solution to 7.0-8.0, fully stirring and uniformly mixing to establish an aqueous two-phase system;
(3) performing double-aqueous phase extraction and microwave extraction: adding bletilla striata powder into a double aqueous phase system to prepare an extraction solution, and carrying out microwave treatment on the extraction solution at 100-600W and 30-60 ℃ for 60-180 min; fully extracting the bletilla polysaccharide, standing for 60-100 min, and centrifuging at 8000-12000 rpm at a high speed to separate a bletilla polysaccharide extract phase;
(4) ultrafiltration, concentration and desalination: performing ultrafiltration desalination on the bletilla striata polysaccharide extract phase obtained in the step (3) by using an ultrafiltration membrane with the molecular weight cutoff of 1-10KD to obtain bletilla striata polysaccharide concentrated solution;
(5) and (3) freeze drying: and (4) freeze-drying the bletilla striata polysaccharide concentrated solution obtained in the step (4) to obtain white uniform bletilla striata polysaccharide solid.
2. The preparation method of bletilla striata polysaccharide according to claim 1, wherein the preparation method comprises the following steps: in the step (2), the low molecular alcohol is C1-C5 straight chain or branched chain alkyl alcohol.
3. The preparation method of bletilla striata polysaccharide according to claim 1, wherein the preparation method comprises the following steps: in the step (2), the inorganic salt is potassium salt, sodium salt or ammonium salt.
4. The preparation method of bletilla striata polysaccharide according to claim 1, wherein the preparation method comprises the following steps: the mass fraction of the inorganic salt is 10-40%, and the mass fraction of the low molecular alcohol is 20-25%.
5. The preparation method of bletilla striata polysaccharide according to claim 1, wherein the preparation method comprises the following steps: in the step (2), the pH value of the aqueous two-phase solution is 7.5.
6. The preparation method of bletilla striata polysaccharide according to claim 1, wherein the preparation method comprises the following steps: in the step (3), the solid-to-liquid ratio of the bletilla striata powder to the double water phase system is 1: 20-1: 100.
7. The preparation method of bletilla striata polysaccharide according to claim 1, wherein the preparation method comprises the following steps: in the step (3), the microwave treatment is carried out for 120min under the microwave treatment condition and the microwave power of 400W at 50 ℃.
8. The preparation method and the application of bletilla striata polysaccharide according to claim 1, wherein the preparation method comprises the following steps: in the step (4), ultrafiltration desalination is carried out by an ultrafiltration membrane with the molecular weight cutoff of 5 KD.
9. The preparation method and the application of bletilla striata polysaccharide according to claim 1, wherein the preparation method comprises the following steps: in the step (5), the average molecular weight distribution of the bletilla striata polysaccharide solid is 1-100 KD; the monosaccharide composition molar ratio n mannose: n-glucose is 4:1 to 1: 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910905352.4A CN110655587B (en) | 2019-09-24 | 2019-09-24 | Preparation method of bletilla striata polysaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910905352.4A CN110655587B (en) | 2019-09-24 | 2019-09-24 | Preparation method of bletilla striata polysaccharide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110655587A true CN110655587A (en) | 2020-01-07 |
| CN110655587B CN110655587B (en) | 2022-06-28 |
Family
ID=69038832
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910905352.4A Active CN110655587B (en) | 2019-09-24 | 2019-09-24 | Preparation method of bletilla striata polysaccharide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110655587B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112724269A (en) * | 2020-12-28 | 2021-04-30 | 吉林化工学院 | Preparation and application of bletilla striata polysaccharide |
| CN113134059A (en) * | 2021-04-29 | 2021-07-20 | 桂林丰润莱生物科技股份有限公司 | Processing method of bletilla striata tablets |
| CN113754791A (en) * | 2021-10-20 | 2021-12-07 | 四川丽妍工坊生物科技有限公司 | Bletilla striata polysaccharide substance and preparation method and application thereof |
| CN115403677A (en) * | 2021-05-28 | 2022-11-29 | 纳米及先进材料研发院有限公司 | Method for extracting polysaccharide |
| CN116178586A (en) * | 2023-03-30 | 2023-05-30 | 金创克(珠海)生物技术有限公司 | Application of bletilla striata polysaccharide in preparation of product with moisturizing effect |
| CN116333183A (en) * | 2023-04-10 | 2023-06-27 | 西南大学 | Method for separating and purifying coptis chinensis polysaccharide by using organic solvent-inorganic salt aqueous two-phase system |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1709915A (en) * | 2005-06-23 | 2005-12-21 | 南京大学医学院附属鼓楼医院 | Polysaccharide material for promoting cell tactophily and preparation thereof |
| CN101899174A (en) * | 2010-08-11 | 2010-12-01 | 珈侬生化科技(中国)有限公司 | Bletilla polysaccharide compound serving as cosmetic raw material and preparation method thereof |
| CN105273094A (en) * | 2015-05-04 | 2016-01-27 | 西华大学 | Method for rapid separation of Ligusticum chuanqiong polysaccharide |
| CN105949340A (en) * | 2016-07-06 | 2016-09-21 | 邵阳学院 | Simultaneous extraction method of radix polygonati officinalis polysaccharide and oyster mushroom polysaccharide |
| CN106866835A (en) * | 2017-04-14 | 2017-06-20 | 张仕英 | A kind of ultrasonic technology extracts the preparation method of bletilla striata extract |
| CN108676107A (en) * | 2018-06-05 | 2018-10-19 | 广东药科大学 | A method of purifying Schizophyllum commune Fr polysaccharides using Bi-aqueous extraction |
-
2019
- 2019-09-24 CN CN201910905352.4A patent/CN110655587B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1709915A (en) * | 2005-06-23 | 2005-12-21 | 南京大学医学院附属鼓楼医院 | Polysaccharide material for promoting cell tactophily and preparation thereof |
| CN101899174A (en) * | 2010-08-11 | 2010-12-01 | 珈侬生化科技(中国)有限公司 | Bletilla polysaccharide compound serving as cosmetic raw material and preparation method thereof |
| CN105273094A (en) * | 2015-05-04 | 2016-01-27 | 西华大学 | Method for rapid separation of Ligusticum chuanqiong polysaccharide |
| CN105949340A (en) * | 2016-07-06 | 2016-09-21 | 邵阳学院 | Simultaneous extraction method of radix polygonati officinalis polysaccharide and oyster mushroom polysaccharide |
| CN106866835A (en) * | 2017-04-14 | 2017-06-20 | 张仕英 | A kind of ultrasonic technology extracts the preparation method of bletilla striata extract |
| CN108676107A (en) * | 2018-06-05 | 2018-10-19 | 广东药科大学 | A method of purifying Schizophyllum commune Fr polysaccharides using Bi-aqueous extraction |
Non-Patent Citations (5)
| Title |
|---|
| CHENG, ZHENYU等: "Simultaneous extraction and purification of polysaccharides from Gentiana scabra Bunge by microwave-assisted ethanol-salt aqueous two-phase system", 《INDUSTRIAL CROPS AND PRODUCTS》 * |
| 吴梧桐主编: "《生物化学》", 31 August 2015, 中国医药科技出版社 * |
| 孔令姗: "白芨多糖的提取与功效研究", 《中国优秀硕士学位论文全文数据库 工程科技Ｉ辑》 * |
| 张卫明等: "白芨多糖胶皮肤毒理学安全性评价研究", 《中国野生植物资源》 * |
| 赵余庆主编: "《中药及天然产物提取制备关键技术》", 31 January 2012, 中国医药科技出版社 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112724269A (en) * | 2020-12-28 | 2021-04-30 | 吉林化工学院 | Preparation and application of bletilla striata polysaccharide |
| CN112724269B (en) * | 2020-12-28 | 2022-07-05 | 吉林化工学院 | Preparation and application of bletilla striata polysaccharide |
| CN113134059A (en) * | 2021-04-29 | 2021-07-20 | 桂林丰润莱生物科技股份有限公司 | Processing method of bletilla striata tablets |
| CN115403677A (en) * | 2021-05-28 | 2022-11-29 | 纳米及先进材料研发院有限公司 | Method for extracting polysaccharide |
| US11780937B2 (en) | 2021-05-28 | 2023-10-10 | Nano And Advanced Materials Institute Limited | Method for extracting polysaccharides |
| CN113754791A (en) * | 2021-10-20 | 2021-12-07 | 四川丽妍工坊生物科技有限公司 | Bletilla striata polysaccharide substance and preparation method and application thereof |
| CN116178586A (en) * | 2023-03-30 | 2023-05-30 | 金创克(珠海)生物技术有限公司 | Application of bletilla striata polysaccharide in preparation of product with moisturizing effect |
| CN116333183A (en) * | 2023-04-10 | 2023-06-27 | 西南大学 | Method for separating and purifying coptis chinensis polysaccharide by using organic solvent-inorganic salt aqueous two-phase system |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110655587B (en) | 2022-06-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110655587B (en) | Preparation method of bletilla striata polysaccharide | |
| Shang et al. | Influences of extraction methods on physicochemical characteristics and activities of Astragalus cicer L. polysaccharides | |
| Wu et al. | Comparison of different extraction methods of polysaccharides from cup plant (Silphium perfoliatum L.) | |
| Xu et al. | Isolation, purification, and antioxidant activities of degraded polysaccharides from Enteromorpha prolifera | |
| Yan et al. | Ultrasonic-assisted extraction optimized by response surface methodology, chemical composition and antioxidant activity of polysaccharides from Tremella mesenterica | |
| Seedevi et al. | Isolation and chemical characteristics of rhamnose enriched polysaccharide from Grateloupia lithophila | |
| CN109651532B (en) | Dendrobium officinale glucomannan | |
| Xiong et al. | Physicochemical properties, antioxidant activities and α-glucosidase inhibitory effects of polysaccharides from Evodiae fructus extracted by different solvents | |
| CN110538112A (en) | Glycyrrhiza glabra extract composition with function of enhancing skin immune barrier and preparation method and application thereof | |
| Yang et al. | Characterization and antioxidant activity of a novel polysaccharide from Pholidota chinensis Lindl. | |
| Cai et al. | Sulfated modification and biological activities of polysaccharides derived from Zizyphus jujuba cv. Jinchangzao | |
| WO2022016644A1 (en) | Acanthopanax senticosus harms homogeneous polysaccharide, preparation method therefor and use thereof | |
| Gu et al. | The structures of two glucomannans from Bletilla formosana and their protective effect on inflammation via inhibiting NF-κB pathway | |
| CN107012184B (en) | Angelica dahurica polysaccharide extracted by enzyme method, preparation method and application thereof | |
| CN110066349B (en) | Low-molecular-weight blackberry polysaccharide and preparation method thereof | |
| CN111410698A (en) | Camel thorn sugar polymer and preparation method and application thereof | |
| CN113698502A (en) | Bletilla striata fibrous root oligosaccharide and preparation method and application thereof | |
| CN115414379B (en) | Application of notoginseng polysaccharide SQP20 in preparation of medicine for treating intestinal injury and inflammatory infiltration | |
| CN112159484B (en) | Anticoagulant fructus polygoni multiflori polysaccharide and extraction and separation method and application thereof | |
| CN115160450A (en) | Rapid preparation method and application of Pholiota nameko polysaccharide | |
| CN115232220A (en) | Ganoderma lucidum polysaccharide and extraction method and application thereof | |
| CN112107590B (en) | Application of swim bladder-derived heparin mucopolysaccharide in preparation of angiogenesis inhibitor | |
| CN115166089A (en) | Method for identifying chlorella pyrenoidosa by using methylated sulfated oligosaccharide group | |
| CN114524887B (en) | Method for separating and characterizing ginseng polysaccharide with pharmaceutical activity function | |
| CN108660175B (en) | A kind of plant source polypeptide and the application in cosmetics |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |